Defective DNA mismatch repair (MMR) causes cancer. MMR mutations use multiple distinct mechanisms to cause cancer: (A) Defective repair of insertion/deletion frameshift mutations (commonly called Microsatellite Instability or MSI), (B) Defective repair of single-basepair DNA mismatches, and (C) Failure to initiate apoptosis in response to DNA damage. Previously, I cloned MLH3. Both inherited and sporadic MLH3 mutations cause CRC and perhaps other types of cancer as well. Gerrnline and sporadic MLH3 mutations are associated with both MSI v and MSI- CRCs. In mice, Mlh3 is essential for meiosis. New results from my lab demonstrate Mlh3 -/- and Mlh3-/-/Pms2-/- mice develop multiple types of cancer, including bowel cancer. However, studies to date have not addressed critical questions: What are the mechanisms of carcinogenesis in cells with MLH3 mutations? What are the mechanisms of carcinogenesis in cells with both MLH3 and PMS2 mutations? We therefore propose the following Aims: Specific Aim 1: To Identify the Mechanisms of Apc Inactivation in Mlh3 -/- and Mlh3-/- Pms2-/- Bowel Cancers. These mechanisms are important to determine because MLH3 and PMS2 mutations underlie both inherited and sporadic forms of CRC in patients. Specific Aim 2: To Analyze Mlh3 -/- Cells for Defects in DNA Damage Induced Apoptosis as a Potential Mechanism of Carcinogenesis. This aim tests the hypothesis that Mlh3 -/- cells do not correctly initiate apoptosis in response to DNA damage as a potential mechanism of carcinogenesis. Specific Aim 3: To Determine Insertion/Deletion MSI and Single-Basepair Mismatch Repair Phenotypes of Mlh3 -/-cells in vitro and in rive as potential mechanisms of carcinogenesis. The aim tests two hypotheses: (1) Mlh3 mutations cause MSI and (2) Mlh3 mutations disrupt single-basepair mismatch repair.